The development of protective or therapeutic HIV vaccines has been hampered by unprecedented challenges, primarily due to the unique properties of the HIV-1 envelope (Env) trimer, a cleverly engineered entry machinery that features an extraordinary assortment of immune-evasion tactics, including antigenic variation, heavy glycosylation of exposed surfaces and conformational camouflage. Further insights into the complex structure-function relationships in the HIV-1 Env trimer and its protective shield will thus be critical to guide the rational design of a protective vaccine. 1) Discovery of a second receptor-binding site in the HIV-1 Env trimer. The CD4-binding site (CD4-BS) has been finely mapped by mutagenesis and co-crystallization of the Env glycoprotein gp120 in complex with soluble CD4 (sCD4). However, most of these studies were performed with monomeric gp120 subunits, thereby hindering the evaluation of the role of quaternary elements assembled in the pre-fusion trimeric structure that may be involved in the initial CD4 interaction. By in silico docking of CD4 to the Env trimer, we found that CD4 appears to make contact not only with the previously defined CD4-BS in the outer domain of a single gp120 protomer, but also with a second CD4-binding site (henceforth defined as CD4-BS2) located in the inner domain of a neighboring protomer. To gain structural insights into the interactions of CD4 with the trimer, we obtained a cryoEM structure of soluble CD4 in complex with a conformationally-constrained soluble trimer, DS-SOSIP.664, which remains in a pre-fusion, closed conformation even after interaction with CD4. The reconstructed cryo-EM structure shows domain 1 of sCD4 inserted between two gp120 protomers and establishing contacts with two adjacent gp120 protomers, confirming our initial model. To elucidate the functional relevance of the quaternary CD4 contact to HIV-1 infection, we mutagenized residues E62, E64, H66, and K207 in full-length gp160 from 6 HIV-1 isolates of different genetic subtypes, namely, BG505 (clade A), BaL and JR-FL (clade B), ZM106.9 (clade C), QD435.A4 (clade D), and CM244 (clade E), and generated infectious viral pseudoparticles for each mutant. Both individual and combined charge inversions were introduced into each Env to create a reverse-charged surface. Strikingly, we found that charge inversions of E62/E64, H66 and K207 virtually abrogated the infectivity of all the HIV-1 Env tested, regardless of their genetic clades. By surface plasmon resonance (SPR) analysis, all CD4-BS2 mutants showed a markedly reduced sCD4-binding affinity, with K207E exhibiting the most pronounced decrease. Next, we studied the events downstream of CD4 binding, which induce gp120 to acquire coreceptor-binding competence. After pre-activation with sCD4, none of the CD4-BS2 mutants were able to bind to the CCR5 coreceptor. To further validate the role of CD4-BS2 in the envelope-receptor interaction, we investigated the CD4 residues that make contact with CD4-BS2. Close examination of the contact site in the fitted model and in the MD simulation revealed four residues in CD4 domain 1 (D1), namely, K21CD4 and K22CD4 in the CDR1-like loop, and D63CD4 and Q64CD4 in the DE loop, as potentially interactive with CD4-BS2. Each residue was mutagenized by charge inversion, and full-length CD4 mutants were expressed on the surface of Cf2Th/syn-CCR5 cells and tested for HIV-1 receptor function. Charge inversion of two CD4 residues, K22CD4 and D63CD4, caused a dramatic loss of HIV-1 receptor function, as seen with both the BG505 and BaL envelopes, while charge inversion of K21CD4 had more limited effects, and alanine substitution of Q64CD4 had no effects. Altogether, these results delineate the initial contact of the HIV-1 Env with the CD4 receptor, documenting the quaternary configuration of the functional HIV-1 receptor-binding site, a feature previously recognized for Picornaviruses, but never hitherto for enveloped viruses. The discovery of a new CD4-binding site paves the way toward the development of new strategies of treatment and vaccine. 2) Selected HIV-neutralizing antibodies mimic the CD4 binding mode and interact with a quaternary site. We also demonstrated by docking and mutagenesis that selected anti-CD4-BS antibodies, such as VRC03, VRC06 and VRC-CH31, mimic the quaternary binding mode of CD4, establishing contacts with two adjacent gp120 protomers. VRC03 and VRC06 belong to the VRC01 class of anti-CD4-BS antibodies, all of which originate from the same germline heavy-chain gene, VH1-2. However, unlike other members of this class, VRC03 and VRC06 contain an extended framework region 3 (FR3) loop in their heavy chain, which was predicted by docking to mediate quaternary contacts. A high-resolution structure of VRC03 in complex with a soluble Env trimer indeed confirmed that the FR3 loop of this antibody interacts with an adjacent gp120 protomer. In contrast, some of the most potent broadly neutralizing antibodies (bNAbs) of the VRC01 class, such as VRC01 itself, VRC07 and N6, appear to interact with a single gp120 protomer, and indeed were insensitive to mutations in CD4-BS2. This work opens the possibility of creating chimeric antibodies bearing the FR3 loop of VRC03 or VRC06 engrafted onto the FR3 domain of some of the most potent bNAbs with the objective of rendering them even more potent against HIV-1. 3) Structure-guided interdomain stabilization of the HIV-1 envelope trimer abrogates CD4 binding and improves immunogenicity. The HIV-1 Env inherent flexibility and susceptibility to antigenic remodeling upon CD4 binding represent key obstacles to the development of a protective vaccine. Hence, stabilization of the trimer in a CD4-resistant conformation should preserve the native antigenic state. we locked the HIV-1 Env in pre-fusion configuration via structure-guided introduction of neo-disulfide bonds bridging the gp120 outer and inner domains, resulting in improved antigenic profile and impaired CD4 binding. This design was successfully applied to both soluble cleaved trimers and native full-length gp160 from different HIV-1 clades. Crystallization of a stabilized trimer illustrated the structural basis for CD4-binding impairment. Immunization of rabbits with locked soluble trimers from two different clades elicited the production of neutralizing antibodies against tier-2 viruses with repaired glycan shield, which was unaffected by exposure to a functional CD4 mimic. Interdomain stabilization provides a template for the design of Env-based HIV-1 vaccines.